Purpose: To investigate the therapeutic potential of 213Bilabeled multiple targeted α-radioimmunoconjugates for treating prostate cancer (CaP) micrometastases in mouse models.

Experimental Design: PC-3 CaP cells were implanted s.c., in the prostate, and intratibially in NODSCID mice. The expression of multiple tumor–associated antigens on tumor xenografts and micrometastases was detected by immunohistochemistry. Targeting vectors were two monoclonal antibodies, and a plasminogen activator inhibitor type 2 that binds to cell surface urokinase plasminogen activator, labeled with 213Bi using standard methodology. In vivo efficacy of multiple α conjugates (MTAT) at different activities was evaluated in these mouse models. Tumor growth was monitored during observations and local regional lymph node metastases were assessed at the end of experiments.

Results: The take rate of PC-3 cells was 100% for each route of injection. The tumor-associated antigens (MUC1, urokinase plasminogen activator, and BLCA-38) were heterogeneously expressed on primary tumors and metastatic cancer clusters at transit. A single i.p. injection of MTAT (test) at high and low doses caused regression of the growth of primary tumors and prevented local lymph node metastases in a concentration-dependent fashion; it also caused cancer cells to undergo necrosis and apoptosis.

Conclusions: Our results suggest that MTAT can impede primary PC-3 CaP growth at three different sites in vivo through induction of apoptosis, and can prevent the spread of cancer cells and target lymph node micrometastases in a concentration-dependent manner. MTAT, by targeting multiple antigens, can overcome heterogeneous antigen expression to kill small CaP cell clusters, thus providing a potent therapy for micrometastases.

Translational Relevance

The control of metastatic prostate cancer (CaP) remains an elusive objective. Multiple targeted α therapy (MTAT) is a combined therapy designed for systemic radiation therapy against cancer cells showing heterogeneous expression of targeted antigens. We have established for the first time the growth of CaP in three different sites in animals, mimicking clinical CaP metastases, and tested the in vivo efficacy of multiple α conjugates (MTAT) at different treatment activities.

Our results indicate that MTAT could regress primary CaP growth s.c., in the prostate, and in the bone, preventing the spread of cancer cells and targeting lymph node micrometastases in a concentration-dependent manner. MTAT may be a potent therapeutic agent against micrometastatic CaP in late stage hormone refractory disease, overcoming the problem of heterogeneous expression of targeted antigens.

The incidence of prostate cancer (CaP) is increasing; the high mortality rate is associated with widespread metastatic disease. Despite surgery and radiation therapy that can treat localized disease and the possibility of early diagnosis through testing for serum prostate-specific antigen, up to 30% of treated CaP patients suffer relapse. Most of these show an initial response to androgen-ablation therapy because early CaP growth is androgen dependent but eventually progress to hormone refractory CaP; this no longer responds to androgen ablation. At that stage, there is no curative therapy for metastatic CaP and median survival is ∼1 year. Nonhormonal agents have been evaluated for patients with hormone refractory CaP but have had limited antitumor activity, an objective response rate of <20% and did not show survival benefit (1). Combined radiation therapy and chemotherapy (2) or radiation and bisphosphonates (3) have shown only limited success in controlling CaP progression. Furthermore, although recent phase II studies with docetaxel (taxotere) display important single agent (4) or combination activity (5) in patients with hormone refractory CaP, the increase in survival is only a median of ∼2 months in ∼40% of patients. As such, metastatic hormone refractory CaP in 2007 remains an incurable disease with a median survival of 18 to 20 months with current docetaxel-based chemotherapy regimens. New, innovative therapies for metastatic CaP are needed.

α-Particle emitters provide an attractive class of radionuclides for targeted radionuclide therapy, particularly for treating minimal residual disease. These α particles, such as Bismuth-213 (213Bi) with a short half-life (t ½ = 46 min), a short range of only a few cell diameters (∼80 μm), and a high linear energy transfer, are more effective than the low linear energy transfer, longer range β particles for destroying cancer cells, and minimizing damage to normal tissues. Therapy using α-particles has been proposed for use in single-cell disorders such as leukemias, lymphoma, and micrometastatic carcinomas (69), where rapid targeting to cancer cells is possible.

The progression of CaP from a hormone-naïve primary to increasingly androgen-independent invasive and metastatic disease is associated with various molecular and genetic changes, which can modulate the expression of cell surface tumor-associated antigens, usually in a heterogeneous fashion. Targeting cancer surface antigens with directed vectors is currently undergoing a resurgence of interest. In the present study, two monoclonal antibodies (MAb; C595 and BLCA-38) and one protein, PAI2, were selected as vectors to deliver the cytotoxic radionuclide, 213Bi for targeted cancer therapy.

MAb C595 is raised against the protein core of human MUC1 (urinary epithelial mucin1). We have shown that MAb C595 binds strongly to three metastatic CaP cell lines (PC-3, DU 145, and LNCaP-LN3; ref. 10), and to over 80% of CaP sections, whereas no staining was found in normal prostate (11). Recently, we have successfully used 213Bi-C595 α conjugates (ACs) to target single monolayer CaP and pancreatic cancer cells in vitro (10, 12), indicating that MUC1 is a useful therapeutic target for CaP therapy.

MAb BLCA-38 recognizes an unknown cell surface and cytoplasmic glycoprotein. We have recently found that BLCA-38 binds with variable intensity to the surface of most human CaP cell lines, and also to CaP tissues (13). 213Bi-BLCA-38 AC used for CaP therapy was found to be cytotoxic to single CaP cells in vitro (10).

Urokinase plasminogen activator (uPA), a member of the serine protease family, is strongly implicated as a promoter of tumor progression in various human malignancies, including CaP (14). Normal, quiescent tissues such as brain and blood express little or undetectable uPA (15, 16). Binding to its receptor (uPAR), uPA efficiently converts the inactive zymogen, plasminogen, into the active serine protease, plasmin, which then directly or indirectly cleaves extracellular matrix components including laminin, fibronectin, fibrin, vitronectin, and collagen (17). Overwhelming evidence shows that the cell surface-associated uPA/uPAR complex is causatively involved in tumor invasion and metastasis of many types of cancers by exerting multifaceted functions via either direct or indirect interactions with integrins, endocytosis receptors, and growth factors.

The activity of uPA is physically regulated by plasminogen activator inhibitors type 1 and 2 (PAI1 and PAI2), and by uPAR. Both PAI1 and PAI2 belong to the serpin (serine protease inhibitor) superfamily and form SDS-stable 1:1 complexes with uPA. A recent study reported that the efficient and rapid inhibition of uPAR-bound uPA by PAI2 at the surface of PC-3 CaP cells leads to the rapid internalization of uPAR/uPA-PAI2 complexes and delivery into endosomes and lysosomes, and that PAI2 binding capacity and internalization is uPA dependent (18). These results suggest that PAI2 could be used as a carrier to specifically deliver cytotoxins to uPA-positive cancer cells. PAI2 can target cancer cell surface uPAR-bound uPA to form an uPA-uPAR-PAI2 complex (19). In earlier studies, we have successfully targeted prostate and breast cancers using 213Bi-PAI2 AC (9, 10, 20, 21).

Despite some success with single agents as described above, and given the highly heterogeneous nature of advanced CaP, we considered that targeting three identified tumor-associated antigens (MUC1, BLCA-38, and uPA) simultaneously on CaP cells with different MAbs or proteins would provide an advantage over targeting of a single antigen to overcome heterogeneous antigen expression. Some cells survive targeting by individual conjugates, frustrating the achievement of a cure. The use of multiple targeting vectors, armed with radionuclides to deliver the cytotoxic agents specifically to cancer cells, provides a valuable alternative approach for cancer therapy. Multiple-targeted α therapy (MTAT) is a combined therapy in which several antibodies/proteins are labeled with 213Bi and administered as a cocktail. In this study, we have used a cocktail of 213Bi-labeled nondirected antibodies and protein as a control rather than 213Bi-labeled individual antibodies or proteins. The number of studies required to examine three different sites of tumor growth in mice would have made the use of individual antibodies/proteins as controls very difficult to perform and a nonspecific control was considered to be suitable.

Antibodies

MAbs/proteins were kindly provided as follows: MAb C595 from A. Perkins (Nottingham University, Nottingham, UK); nonspecific isotype control IgG1 MAb, (A2, against mineral oil plasmacytoma) by A. Collins (University of New South Wales, Sydney, NSW, Australia); MAb BLCA-38 by Minomic Pty Ltd; human recombinant PAI2 (47 kDa), reactive with membrane-bound uPA, by PAI2 Pty Ltd. Other MAbs used were mouse MAb #394 IgG1 (anti-uPA; American Diagnostica, Inc.), polyclonal rabbit antimouse IgG/biotinylated, streptavidin/horseradish peroxidase, and mouse IgG1–negative control MAb from Dakopatts. Mouse antihuman IgG3-negative control MAb was purchased from Zymed Laboratories, Inc.

Cell lines and cell culture

The androgen-independent PC-3 CaP cell line (American Type Culture Collection) was cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 50 units/mL penicillin, and 50 units/mL streptomycin. All tissue culture reagents were supplied by Invitrogen Australia Pty Ltd unless otherwise stated. PC-3 cells were maintained in a humidified incubator at 37°C and 5% CO2. Cells were harvested, washed, and resuspended in Dulbecco's PBS at 4°C until used for animal inoculation (0-2 h).

Animal models

Male, 6- to 8-wk-old NODSCID mice (Animal Resources Centre) were housed and maintained in laminar flow cabinets under specific pathogen-free conditions in facilities approved by the University of New South Wales Animal Care and Ethics Committee in accordance with the regulations and standards approved by US Department of Defense. Mice were kept at least 1 wk before experimental manipulation. Anesthesia was done using a Sleep Easy anaesthetic machine (with 5% isoflurane for induction and 3% for maintenance, with oxygen at 3 liters/min). Before radiography, mice were anesthetized by 50 mg/kg ketamine and 5 mg/kg xylazine i.p. All mice remained healthy and active during the experiment.

S.c. xenografts

As described previously (20), cultured PC-3 cells (1.5 × 106/injection) in 100 μL Dulbecco's PBS were implanted s.c. in the right rear flank region of NODSCID mice. Tumor progression was documented once weekly by measurements using calipers, and tumor volumes were calculated by the following formula: length × width × height × 0.52 in millimeters (22) for up to 8 wk. Upon sacrifice, local regional lymph nodes were removed for histologic examination.

Intraprostate orthotopic model

Viable PC-3 cells (1 × 106/injection) in 50 μL of DBPS were injected into the prostatic lobe at the base of the seminal vesicles after exposure through a lower midline laparotomy incision as previously described (23). Mice were followed for 8 wk. Upon sacrifice, local regional lymph nodes were removed for histologic examination.

Intratibial CaP model

As described (24, 25), PC-3 CaP cells (5 × 104/20 μL) were injected from a 1 mL syringe using a 26-gauge needle. Mice were monitored twice weekly for up to 6 wk.

Radiographic examination

Radiographs taken with a Faxitron (Model# MX-20, Serial#2321A0509, Option#Ao7; 50-60Hz, 150 VaMax; 60090; 32 kV, 2.3 s, 20.7 mA/s) were done on the tibiae of mice injected with PC-3 cells 1, 2, 4, and 6 wk postinoculation. These were quantified visually using a 10× Deluxe loop objective by two observers; images were further processed in Adobe Photoshop Elements 4.0.

Conjugation of bifunctional chelate to MAbs or proteins and radiolabeling

213Bi was eluted from a 225Ac/213Bi generator (Institute for Transuranium Elements) using 600 μL of freshly prepared 0.1 mol/L NaI/0.1 mol/L HCl as the (BiI4)/(BiI5)2− anion species, neutralized to pH 4 to 4.5 using 3M ammonium acetate, and immediately used to radiolabel MAb constructs (10, 26). A time of 2 to 3 h was allowed for 213Bi to grow back in the generator for the next elution. The labeling procedure for MAbs has been described previously (10) and used purified chelator, cyclic diethylenetriaminepentacetic acid anhydride (cDTPA; Sigma-Aldrich Pty, Limited) at a chelator/antibody molar ration of 20:1 (45 min on ice). Bovine serum albumin (BSA) was used as a nonspecific protein control for PAI2 binding. PAI2 and BSA, adjusted to pH 8.2, were conjugated with cDTPA, as described (10). α Conjugates of each antibody and protein were obtained by labeling with free 213Bi for 20 min at room temperature, providing a radiolabeling efficiency, determined by instant TLC of 80% to 95% depending on the vector. The absolute activities of α conjugates were corrected by reference to a secondary standard supplied by ITU. All MTAT, control cocktail (CC), and cold control preparations were adjusted to a final volume of 200 μL using the buffer control [0.06 mol/L HI, 0.2 mol/L citrate buffer (pH 5.5), and 1× PBS].

Treatment protocols

Toxicity studies in NODSCID mice without tumors. Toxicity studies were done to determine the maximum tolerance dose in mice for MTAT and CCs (Supplementary Fig. S1). The dose-tolerance relationship was examined in NODSCID mice (without tumors) for a single i.p. administration of MTAT compared with control treatments. Groups of 5 mice received a total injected activity of 237, 355, 474, 592, and 710 MBq/kg of MTAT, as well as 474, 592, and 710 MBq/kg of nonspecific CC, cold control (cDTPA labeled with C595, BLCA-38, and PAI2, each 33% of total amount, concentration 20 mg/kg) and saline (the same volumes). Mouse weights were compared with those at day 0 (first day of treatment administration) to determine percentage weight change. The dose-limiting toxicity was defined as end points: 15% loss of body weight or distressed behavior (i.e., loss of appetite and activity, hunched posture). The maximum tolerance dose was defined as the highest dose at which one third of the cohort reached dose-limiting toxicity end points (27). After 13 wk, healthy mice were euthanized. The following experiments were based on the maximum tolerance dose from the toxicity studies.

Efficacy studies in three CaP animal models. Efficacy studies of MTAT in mice that received s.c., orthotopic, or intratibially injected PC-3 cells were done at 14 or 7 d post–cell inoculation, respectively (Supplementary Fig. S1). Four groups of 8 mice that received a single i.p. injection of multiple ACs (test) at 296 or 592 MBq/kg, CC (nonspecific ACs) at 592 MBq/kg, and equal volumes of saline. For s.c. and orthotopic models, 3 mice per group were sacrificed after 7 d (21 d post–cell inoculation), whereas for intratibial model, sacrifice for all mice was after 6 wk. After 8 wk, the remaining mice were euthanized. Tumors were assessed for histologic change and for analysis of apoptosis.

Mouse tissues and histology

All tissues were formalin fixed and paraffin embedded, then whole-mount embedded. H&E-stained sections were reviewed to assess tumor development.

For toxicity studies, important mouse organs (kidney, liver, heart, and bone marrow) were collected for pathologic examination (IDEXX Laboratories). For s.c. and orthotopic models, tumor xenografts and para-aortic lymph nodes were collected at the end of experiments or 7 days after treatment and processed for histology, immunohistochemistry, and terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling (TUNEL) assay. For the intratibial model, the hind limbs were immediately dissected at euthanasia and fixed in 10% neutral buffered formalin for 24 h. After fixation, the limbs were removed from most of the muscle tissue and transferred to a sterile decalcification solution [10% EDTA and 0.5% paraformaldehyde in PBS (pH 8.0)] for 2 wk, with the solution changed every 2nd day. Samples were then paraffin embedded for histology, immunohistochemistry, and TUNEL assay.

Hematologic toxicity and renal function examination

To determine hematologic toxicity, 200 μL of blood in each mouse were collected into K3 EDTA and Z serum gel minicollect tubes (Greiner Bio-one) via saphenous vein by Microvette (SARSTEDT) before treatment and at 2 and 3 wk post-MTAT or control conjugates injection. Hematologic analyses of WBC, lymphocytes, RBC, and platelet counts were done. Blood was obtained at the end of experiment for biochemical analysis of serum for renal functions.

Immunohistochemistry

Standard immunoperoxidase procedures were used for MUC1, uPA, and BLCA-38 expression. Briefly, paraffin sections were deparaffinized in xylene, followed by a graded series of alcohols (100%, 95%, and 75%) and rehydrated in TBS (pH 7.5). Slides were subsequently immersed in boiling 0.01 mol/L citrate buffer (pH 6.0) for 15 min to enhance antigen retrieval, treated with 3% hydrogen peroxidase, and then incubated with primary MAbs o/n at 4°C. Antibodies were used at dilutions of 1:500 for MUC1, 1:20 for #394 (anti-uPA), and 1:200 for BLCA-38, respectively. After washing with TBS, slides were incubated with rabbit anti-mouse biotinylated IgG second antibody (1:300 dilution) for 45 min at room temperature, and then with avidin/biotinylated horseradish peroxidase solution (1:300 dilution) for 30 min at room temperature. Positive cells appear brown. Sections were finally developed with 3, 3′ diaminobenzidine substrate solution, then counterstained with hematoxylin. Control slides were treated in an identical manner, and comprised the isotype MAb or omission of the primary antibody as a negative control. Positive controls were chosen depending on different MAbs, colon carcinoma tissue for MUC1; PC-3 CaP cell line for uPA; DU145 CaP cell line for BLCA-38.

Assessment of immunostaining

Staining intensity (0-3) was assessed using light microscopy (Leica microscope) at a ×40 objective as - (negative), + (weak), ++ (moderate), and +++ (strong). Evaluation of tissue staining was done, independently, by two experienced observers (YL and ES). All specimens were scored blind and an average of grades was taken. If discordant results were obtained, differences were resolved by joint review and consultation with a third observer, experienced in immunohistochemical pathology.

TUNEL assay for apoptotic cells in vivo

To investigate the possible mechanism of cell death after MTAT, TUNEL assay was done using a published method (28) with the TdT-fragEL in situ apoptotic detection kit according to the manufacturer's instruction (Oncogene Research Products), using treated HL60 cells as a positive control. Cells were examined using a Leica light microscope.

Statistical analysis

All numerical data were expressed as the average of the values obtained, and the SD was calculated. Data from treated and control groups were compared using the two-tail Student's t test. All P values were two-sided. One way ANOVA, followed by the Dunnett's post hoc test, was done to determine the significance of differences between the means in s.c. model of tumor volume changes. P value of <0.05 was considered significant. All statistical analyses were done using the GraphPad Prism 4.00 package (GraphPad).

Expression of target antigens in primary PC-3 xenografts and micrometastases. Target antigens were expressed heterogeneously on CaP xenografts and cancer cell clusters (CCCs) at transit in lymphatic vessels (Fig. 1). Immunohistochemical staining of tumor xenografts and CCCs with #394 MAb (anti-uPA) showed strong immunoreactivity in xenografts grown s.c. (Fig. 1A), in the prostate (Fig. 1E), or intratibially (Fig. 1I), and weak immunoreactivity in CCCs (Fig. 1M). Staining for MUC1 was strong in tumors grown s.c. (Fig. 1B), moderate in those grown in prostate (Fig. 1F), strong in intratibial tumors (Fig. 1J) with moderate immunoreactivity in CCCs (Fig. 1N). The BLCA-38 MAb immunoreactivity was strong in xenografts at all three sites (Fig. 1C, G, and K) and weak in CCCs (Fig. 1O). No staining was seen in xenografts from each site (Fig. 1D, H, and L) or in CCCs (Fig. 1P) with isotype-matched control MAbs. The percentage of positive staining cells in all three tumor xenografts and CCC were 66% to 84% for uPA, 65% to 92% for MUC1, and 60% to 90% for BLCA-38, respectively. The staining results are summarized in Table 1.

Fig. 1.

Heterogenous expression of tumor antigens (uPA, MUC1, and BLCA-38) in PC-3 xenograft tumors from three animal models and cancer cell clusters (CCCs) at transit in lymphatic vessels by immunohistochemistry. Representative images of primary tumors of three animal models and cancer cell clusters showing immunolabeling with #394, C595, and BLCA-38 MAbs. Heterogeneous expression of uPA, MUC1, and BLCA-38 is found in s.c. xenografts (A-C), orthotopic xenografts (E-G), tibial xenografts (I-K), and CCCs at transit in lymphatic vessels (M-O). No staining is found in control tumor sections (D, H, L, and P) without primary MAbs. Brown, positive; blue, nuclear. All photographs ×40 original magnification.

Fig. 1.

Heterogenous expression of tumor antigens (uPA, MUC1, and BLCA-38) in PC-3 xenograft tumors from three animal models and cancer cell clusters (CCCs) at transit in lymphatic vessels by immunohistochemistry. Representative images of primary tumors of three animal models and cancer cell clusters showing immunolabeling with #394, C595, and BLCA-38 MAbs. Heterogeneous expression of uPA, MUC1, and BLCA-38 is found in s.c. xenografts (A-C), orthotopic xenografts (E-G), tibial xenografts (I-K), and CCCs at transit in lymphatic vessels (M-O). No staining is found in control tumor sections (D, H, L, and P) without primary MAbs. Brown, positive; blue, nuclear. All photographs ×40 original magnification.

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Table 1.

The intensity of immunocytochemical staining of uPA, MUC1, and BLCA-38 with #394, C595, and BLCA-38 MAbs in three CaP xenografts and cancer clusters

TumorMAb
#394
C595
BLCA-38
(n = 5)AntigenUPAMUC1BLCA-38A (NK)
sc model  +++ +++ +++ 
  70–75% 85–90% 76–80% 
Orthotopic model  +++ ++ ++ 
  80–84% 65–69% 85–90% 
Intratibial model  +++ +++ +++ 
  68–74% 86–92% 77–81% 
Cancer clusters  +−++ ++ ++ 
  66–70% 76–84% 60–68% 
TumorMAb
#394
C595
BLCA-38
(n = 5)AntigenUPAMUC1BLCA-38A (NK)
sc model  +++ +++ +++ 
  70–75% 85–90% 76–80% 
Orthotopic model  +++ ++ ++ 
  80–84% 65–69% 85–90% 
Intratibial model  +++ +++ +++ 
  68–74% 86–92% 77–81% 
Cancer clusters  +−++ ++ ++ 
  66–70% 76–84% 60–68% 

NOTE: All sections were prepared at the end of experiments.

Abbreviations: −, negative; +, weak; ++, moderate; +++, strong.

Toxicologic evaluation of MTAT and CC. Single-dose administration of MTAT at 237, 355, 474, and 592 MBq/kg did not reach toxicity end points (Fig. 2A). A small acute weight loss of 3% to 8% from day 0 was observed in all cohorts, but there was quick recovery by day 14 with all mice gaining weight thereafter. A single administration of CC at 474 and 592 MBq/kg did not reach toxicity end points (Fig. 2B), whereas a dose of 710 MBq/kg of either resulted in 3 of 5 of mice losing 15% weight loss (Fig. 2A and B). Mice in the control groups (saline and cold control), or receiving MTAT and CC groups (237-592 MBq/kg), were monitored for 90 days posttreatment with no signs of delayed toxicity being observed (Fig. 2A and B). There were no macroscopic signs of chronic toxicity to major organs from any mice. Leukocyte counts were depressed in peripheral blood in treated mice at 2 weeks postinjection, with recovery occurring by 3 weeks, and normal hematology was seen at 90 days. These results suggest that MTAT or CC could cause temporary myelosuppression in bone marrow. Mice treated with 710 MBq/kg of MTAT or CC were euthanized 4 weeks posttreatment because of signs of distress; histology indicated mild radiation nephropathy in these groups. The results suggest the maximum tolerance dose for a single administration of MTAT lies between 592 and 710 MBq/kg. In the ensuing efficacy studies, we chose 592 MBq/kg as high dose and 296 MBq/kg as low dose for the treatment of xenografted mice bearing tumors in 3 different locations.

Fig. 2.

Dose-tolerance studies for escalating single-dose administration of MTAT and CC (nonspecific α-conjugates) in NODSCID mice without tumors. Average percentage weight changes compared with day 0 (i.e., day of treatment). A, dose-tolerance relationship in mice by MTAT. ▪, MTAT (237 MBq/kg); ▴, MATA (355 MBq/kg); ▾, MTAT (474 MBq/kg); ♦, MTAT (592 MBq/kg); •, MTAT (710 MBq/kg). B, dose-tolerance relationship in mice by controls. ▪, saline; ▴, cold control (cDTPA labeled with C595, BLCA-38, and PAI2, each 33% of total amount, concentration is 20 mg/kg); ▾, nonspecific CC (474 MBq/kg); ♦, nonspecific CC (592 MBq/kg); •, nonspecific CC (710 MBq/kg). Points, mean (n = 5 in each group); bar, SD.

Fig. 2.

Dose-tolerance studies for escalating single-dose administration of MTAT and CC (nonspecific α-conjugates) in NODSCID mice without tumors. Average percentage weight changes compared with day 0 (i.e., day of treatment). A, dose-tolerance relationship in mice by MTAT. ▪, MTAT (237 MBq/kg); ▴, MATA (355 MBq/kg); ▾, MTAT (474 MBq/kg); ♦, MTAT (592 MBq/kg); •, MTAT (710 MBq/kg). B, dose-tolerance relationship in mice by controls. ▪, saline; ▴, cold control (cDTPA labeled with C595, BLCA-38, and PAI2, each 33% of total amount, concentration is 20 mg/kg); ▾, nonspecific CC (474 MBq/kg); ♦, nonspecific CC (592 MBq/kg); •, nonspecific CC (710 MBq/kg). Points, mean (n = 5 in each group); bar, SD.

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Effect of MTAT on tumor growth inhibition and development of lymph node metastases from s.c. xenografts. We compared the antitumor activity of MTAT given as a single i.p. injection 14 days posttumor cell inoculation at 296 and 592 MBq/kg/mouse with an equivalent high dose (592 MBq/kg) of CC (hot control). This was made up of one-third of total activity of 213Bi-BSA and two-thirds of total activity of 213Bi-A2 or the same volume of saline. As shown in Fig. 3A, the growth curves of PC-3 s.c. xenografts show incomplete regression after 296 and 592 MBq/kg of MTAT (test). No tumor regression was found in saline and nonspecific CC-treated groups. The regrowth curves for groups treated with MTAT (296 and 592 MBq/kg) were significantly different from those of control groups (P < 0.01). Eight weeks after treatment, MTAT had significantly inhibited PC-3 tumor volumes (35 ± 9 mm3 in mice given 592 MBq/kg-MTAT, 75 ± 11 mm3 in mice receiving 296 MBq/kg-MTAT versus 397 ± 52 mm3 in mice treated with saline; 90% and 80% reductions, P < 0.01). Tumor volumes at the end of these experiments are shown in Fig. 3B.

Fig. 3.

In vivo efficacy of MTAT in s.c. and orthotopic PC-3 xenografts. Tumor volume changes after single MTAT treatment with different activities and controls (A). The mice were treated 10 days postcell inoculation. The tumor volumes in treated groups with MTAT (296 or 592 MBq/kg) show significant decreases compared with those in control groups (P < 0.05), whereas the tumor volumes in MTAT-treated (296 MBq/kg) mice are also significantly different from those given high dose MTAT (592 MBq/kg; P < 0.05). Tumor weight changes after single MTAT treatment with different activities and controls at the end of experiments (B). The mice were treated 10 days postcell inoculation. The tumor weights in CC (H), MTAT (L), and MTAT (H) were lower than those in mice given saline. The tumor weight in MTAT (L) and MTAT (H) were significantly lower than that in control groups (P < 0.05). Representative images of mouse tumor weight changes in mice given s.c. PC-3 tumors (C) and lymph node metastases in mice given intraprostatic PC-3 cell injection (D) and treated with control conjugates or MTAT at the end of experiments. CC (hot control) includes one-third of total activity of 213Bi-BSA and two-thirds of total activity of 213Bi-A2, whereas MTAT includes 213Bi-PAI2, 213Bi-C595, and 213Bi-BLCA-38 (each one-third of total activity). H, high activity of ACs (592 MBq/kg). L, low activity of ACs (296 MBq/kg).

Fig. 3.

In vivo efficacy of MTAT in s.c. and orthotopic PC-3 xenografts. Tumor volume changes after single MTAT treatment with different activities and controls (A). The mice were treated 10 days postcell inoculation. The tumor volumes in treated groups with MTAT (296 or 592 MBq/kg) show significant decreases compared with those in control groups (P < 0.05), whereas the tumor volumes in MTAT-treated (296 MBq/kg) mice are also significantly different from those given high dose MTAT (592 MBq/kg; P < 0.05). Tumor weight changes after single MTAT treatment with different activities and controls at the end of experiments (B). The mice were treated 10 days postcell inoculation. The tumor weights in CC (H), MTAT (L), and MTAT (H) were lower than those in mice given saline. The tumor weight in MTAT (L) and MTAT (H) were significantly lower than that in control groups (P < 0.05). Representative images of mouse tumor weight changes in mice given s.c. PC-3 tumors (C) and lymph node metastases in mice given intraprostatic PC-3 cell injection (D) and treated with control conjugates or MTAT at the end of experiments. CC (hot control) includes one-third of total activity of 213Bi-BSA and two-thirds of total activity of 213Bi-A2, whereas MTAT includes 213Bi-PAI2, 213Bi-C595, and 213Bi-BLCA-38 (each one-third of total activity). H, high activity of ACs (592 MBq/kg). L, low activity of ACs (296 MBq/kg).

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All mice displayed activity during observation; they were sacrificed 8 weeks postcell inoculation when most mice from control groups had metastases in regional lymph nodes, as assessed by H&E staining under light microscopy; metastases were reduced to ∼40% (2 of 5) in mice given 296 MBq/kg MTAT and 0% in those receiving 592 MBq/kg MTAT (see Supplementary Table S1).

Effect of MTAT on tumor growth inhibition and development of lymph node metastases in mice with intraprostatic xenografts. A single i.p. injection at 14 days post–cell inoculation was given to mice carrying orthotopic PC-3 tumors at 296 and 592 MBq/kg/mouse for MTAT, and 592 MBq/kg/mouse for nonspecific CC and saline control groups. Orthotopic tumor growth in the prostate could not be observed by weight changes over time in these mice. We therefore evaluated the tumor weight changes in each group at the end of the experiments (8 weeks after treatment). At this time MTAT (test) significantly inhibited PC-3 tumor weight (200 ± 50 mg in 592 MBq/kg–treated group and 300 ± 50 mg in 296 MBq/kg–treated group versus 500 ± 100 mg and 400 ± 100 mg in mice receiving saline or nonspecific CC, respectively). This represented 60% and 40% reductions in MTAT-treated versus saline control or CC groups (P < 0.05). Tumor weight changes at the 8-week time point are shown in Fig. 3C.

At this time, mice in different groups were sacrificed and local lymph nodes were examined for metastases by H&E staining. All mice from control groups developed regional lymph node metastases, compared with 20% (1 of 5) in those treated at 296 MBq/kg and 0% after treatment at 592 MBq/kg (see Supplementary Table S1). The number of lymph node metastases in control groups (2-3) was higher than that in mice given MTAT (0-1). Tumor volumes of MTAT-treated mice were smaller than those in control groups. Typical pictures are shown in Fig. 3D.

Effect of MTAT on inhibition of intratibial tumor growth. Seven days postintratibial PC-3 cell inoculation, groups of mice were treated i.p. with a single injection of 296 or 592 MBq/kg/mouse of MTAT, 592 MBq/kg/mouse of nonspecific CC or saline. Intratibial tumor growth was monitored by radiographic examination. After 1 week, intratibial tumors were observed in 6 of 32 (18%) of the mice. From 2 weeks onward, tumors were observed in 16 of 16 of tibiae inoculated in control groups, and 4 of 8 and 3 of 8 in mice treated at 296 and 592 MBq/kg/mouse of MTAT, respectively. After 2 weeks, 1 more tumor appeared in mice given MTAT at low dose (296 MBq/kg) and high dose (592 MBq/kg; see Supplementary Table S2). The lesions showed radiographic features typical of human primary osteosarcoma, including metaphyseal osteolysis and periosteal elevation, with new bone formation and extension into the surrounding soft tissue. Typical pictures for tumor progression in bone are shown in Fig. 4. The lesions in control mice were small at 2 weeks (Fig. 4A and B) and became bigger by 6 weeks (Fig. 4E and F). Only 38% to 50% of MTAT-treated mice showed the appearance of tibial damage by 2 weeks, whereas no tumor lesions were found in most of treated mice (Fig. 4C and D). By 6 weeks after treatment, tumor lesions in the MTAT-treated group (high dose) were obviously different from those in control groups (P < 0.05; Fig. 4E and F); changes in the tumor lesions after MTAT are summarized in Supplementary Table S3. These results suggest that MTAT could target micrometastases and inhibit metastatic CaP progression to bone.

Fig. 4.

Representative X-ray images of tibia from mice injected with PC-3 CaP cells and treated with CC and MTAT. High activity of MTAT was able to regress PC-3 cell growth with partial regression 1 wk after treatment (D) and subsequent complete regression for up to 6 wk (H). Low activity MTAT caused only partial regression tumor growth (C and G). Both saline and high activity of CC did not prevent tumor development (A and E, and B and F). CC (hot control) includes one-third of total activity of 213Bi-BSA and two-thirds of total activity of 213Bi-A2, whereas MTAT includes 213Bi-PAI2, 213Bi-C595, and 213Bi-BLCA-38 (each one-third of total activity). The semiquantitation scoring was as follows: 0, no lesions; 1, minor change; 2, small lesions; 3, significant lesions. A (1), B (1), C (0), D (0), E (4), F (3), G (2), and H (0). Arrows, areas that are representative of tumor progress in the tibia metaphysis with increasing destructiveness and a growth soft tissue compartment.

Fig. 4.

Representative X-ray images of tibia from mice injected with PC-3 CaP cells and treated with CC and MTAT. High activity of MTAT was able to regress PC-3 cell growth with partial regression 1 wk after treatment (D) and subsequent complete regression for up to 6 wk (H). Low activity MTAT caused only partial regression tumor growth (C and G). Both saline and high activity of CC did not prevent tumor development (A and E, and B and F). CC (hot control) includes one-third of total activity of 213Bi-BSA and two-thirds of total activity of 213Bi-A2, whereas MTAT includes 213Bi-PAI2, 213Bi-C595, and 213Bi-BLCA-38 (each one-third of total activity). The semiquantitation scoring was as follows: 0, no lesions; 1, minor change; 2, small lesions; 3, significant lesions. A (1), B (1), C (0), D (0), E (4), F (3), G (2), and H (0). Arrows, areas that are representative of tumor progress in the tibia metaphysis with increasing destructiveness and a growth soft tissue compartment.

Close modal

Histologic alterations in tumor xenografts after MTAT. To compare the cellular content of each tumor, we harvested tumors from treated and control groups at the end of the experiments and stained them with H&E. As assessed by light microscopy (Fig. 5), some targeted lesions (592 MBq/kg of MTAT from different time points) from each site were found to be composed primarily of acellular material (Fig. 5A, C, and E). Cells were most commonly found in small islands (Fig. 5C) with few blood vessels. In contrast, tumors from mice given nonspecific CC consisted of tightly packed cells, with obvious blood vessels (Fig. 5G, I, and K). The results indicate a large difference in tumor cell burden between MTAT-treated and control mice.

Fig. 5.

Representative images of targeted lesions and TUNEL assays in three CaP animal xenograft models. A to F, targeted lesions; G to L, nontargeted lesions. TUNEL assay shows nuclear chromatin condensation and fragmentation (arrows) in some cells in targeted lesions 36 h after treatment with a 592 MBq/kg MTAT (B, D, and F). The tumors treated with nonspecific CC show normal structure (H, J, and L). Brown, apoptotic cells. MTAT includes 213Bi-PAI2, 213Bi-C595, and 213Bi-BLCA-38 (each one-third of total activity). CC includes one-third of total activity of 213Bi-BSA and two-thirds of total activity of 213Bi-A2. Magnification is ×20 in A, C, E, and K; Magnification is ×40 in B, D, F, G, H, I, J, and L.

Fig. 5.

Representative images of targeted lesions and TUNEL assays in three CaP animal xenograft models. A to F, targeted lesions; G to L, nontargeted lesions. TUNEL assay shows nuclear chromatin condensation and fragmentation (arrows) in some cells in targeted lesions 36 h after treatment with a 592 MBq/kg MTAT (B, D, and F). The tumors treated with nonspecific CC show normal structure (H, J, and L). Brown, apoptotic cells. MTAT includes 213Bi-PAI2, 213Bi-C595, and 213Bi-BLCA-38 (each one-third of total activity). CC includes one-third of total activity of 213Bi-BSA and two-thirds of total activity of 213Bi-A2. Magnification is ×20 in A, C, E, and K; Magnification is ×40 in B, D, F, G, H, I, J, and L.

Close modal

Assessment of cell death after MTAT. To investigate if the mechanisms involved in the induction of apoptosis in targeted lesions of primary tumor xenografts represented a phenotypic response of PC-3 tumors, the TUNEL assay was done. The free 3′ ends generated by apoptotic DNA cleavage were detected by TUNEL assay, in which the nonapoptotic cells stained gray and apoptotic cells stained dark brown. Representative results are shown in Fig. 5. After treatment, MTAT-treated tumor cells from s.c. (Fig. 5B), orthotopic (Fig. 5D), and intratibial sites (Fig. 5F) showed typical apoptotic cell morphology with nuclear chromatin condensation and fragmentation, whereas tumors treated with nonspecific CC did not (Fig. 5H, J, and L). Apoptotic nuclei in the tumors were scattered and located in the targeted lesions. Central necrosis was observed in all tumors; some distinct apoptotic nuclei were observed in these areas.

Metastasis is the principle cause of death in CaP patients. The dissemination of CaP including androgen-dependent and hormone refractory CaP cells is through lymphatics and blood vessels. Once regional lymph nodes become involved in prostate carcinoma, 85% of patients develop distant metastases within 5 years (29). In advanced stage disease, small cancer cell clusters (micrometastases) grow in marrow and bone and are difficult to cure. Due to the heterogeneous nature of metastatic cancer cells, multiple targeting has the potential to increase the likelihood of killing all cancer cells. In the present study, we investigated whether conjugation of 213Bi to different vectors, used together in a cocktail (MTAT), would be useful against CaP cells that express different levels of the target antigens in animal models that mimic clinical metastatic CaP sites in humans.

The present system relies on the targeting of 213Bi to tumor-associated antigen–positive tumor cells. As a result, a much greater fraction of the total energy is deposited in cells with α and very few nuclear hits are required to kill a cell. Our previous studies have shown that antigens (uPA, MUC1, and BLCA-38) are expressed on the surface of single monolayer CaP cell lines including PC-3 (10), as well as by CaP spheroids from DU 145 and LNCaP-LN3 cell lines and frozen human CaP sections (9), whereas uPA is expressed on PC-3 s.c. xenografts and lymph node metastases (20). In this study, we first showed that heterogeneous overexpression of uPA, MUC1, and BLCA-38 was found in PC-3 tumor xenografts implanted in three different sites (s.c., orthotopically, and intratibially) and on metastatic cancer clusters at transit in lymphatics. These results suggest that cancer clones, which escape from primary tumors, maintain expression of the tumor antigens, (uPA, MUC1, and BLCA-38) and these antigens may play an important role in CaP metastases. The heterogeneous overexpression of such antigens makes MTAT a feasible approach to therapy.

Metastases derived from s.c. human CaP xenografts of the PC-3 cell line have been reported (30) and we have described the development of CaP lymph node metastases in 100% of nude mice injected with PC-3 cell lines after 8 weeks (20). In this study, the therapeutic experiments using s.c. xenografts were designed to evaluate the antitumor activity and antimetastatic effect of MTAT administered 14 days posttumor cell inoculation (tumor volume, 35 mm3). Our findings indicate that a single systemic (i.p.) injection of MTAT (test) at doses of 296 and 592 MBq/kg strongly regressed tumor progression for at least 5 weeks. Tumor volumes in the MTAT-treated groups decreased by 90 (high dose) and 80% (low dose) compared with the controls. A single i.p. injection of MTAT at 592 MBq/kg can also completely prevent lymph node metastases for at least 8 weeks, during which period all control mice developed lymph node metastases. The regression of metastatic growth was dose dependent (see Supplementary Table S1). These results suggest that an effective dose of MTAT can regress the growth of small, solid PC-3 tumors but cannot eradicate them completely. This may be because the tumor size affects the efficacy of MTAT. It was reported that radioimmunoconjugate uptake usually decreases due to increased interstitial pressure (resulting from the lack of functioning lymphatic vessels) and restricted blood supply (31). The elevated interstitial pressure may also act as a physiologic barrier to the delivery of MTAT by preventing extravasation of MAbs C595, BLCA-38, and PAI2. A possible explanation for preventing lymph node metastases could be that after absorption through the peritoneal vascular system, an effective dose of MTAT can target PC-3 cancer cells at transit or micrometastases in lymph nodes that express uPA, MUC1, and BLCA-38, leading to their complete regression. McDevitt et al. (7) reported that 213Bi-J591 (anti-PSMA) can regress the growth of LNCaP spheroids comprising ∼1,000 cancer cells in vitro. Similarly, our recent study indicates that MTAT can completely target CaP spheroids (∼100 μm) in a dose-dependent manner (9). These in vitro results also support our in vivo targeting results.

More realistic models of human CaP can be achieved by orthotopic implantation (32). Rembrink et al. (33) injected suspensions of PC-3 cells into the nude mouse prostate, and lymph node metastases were found in almost all animals. Compared with s.c. xenografts, intraprostatic tumors cannot be monitored by changes in tumor volume change overtime, necessitating assessment of tumor volumes/weight after euthanasia of mice. In the present study, this was done in orthotopic tumor-bearing mice at the end of experiments. The inhibition of tumor progression for at least 5 weeks by a single i.p. injection of MTAT at doses of 296 (40% inhibition) and 592 MBq/kg (60% inhibition) compared with control saline-treated mice was also accompanied by total inhibition of lymph node metastases (high dose) and 80% inhibition (low dose) for at least 8 weeks. Whereas primary tumors in control mice grew to 15 mm in diameter (when they had to be euthanized), those in MTAT-treated mice remained smaller (6-10 mm; Fig. 3C). These results indicate that a dose-dependent effect of MTAT was seen against primary and secondary PC-3 tumor growth both s.c. and orthotopically.

In the present study, we chose 14 days postinoculation as our treatment point for s.c. and orthotopic xenografts. Based on our previous results, small nodules with PC-3 cells could be vascularized at 3 days post–cell inoculation in nude mice and nodules with blood vessels could be formed at 10 days post–cell inoculation (34). At this time point, PC-3 CaP tumors may have a potential to metastasize. Given from our previous study that the cytotoxicity of MTAT to CaP spheroids is highly dependent on antigenic expression, concentration of radioactivity, and spheroid size (9), and it is possible that MTAT given i.p. at this time point can target micrometastases in regional lymph nodes, cancer cell clusters at transit, or small tumor nodules and eradicate cancer cells expressing heterogeneous tumor antigens.

A major clinical complication in patients with advanced CaP is metastasis to bone (35, 36). The effect of this problem is evident from its occurrence in 30% of men newly diagnosed with CaP and up to 75% of patients at some time during their illness (35, 36). Depending on the predominant molecular pathways at play, these secondary tumors can cause either osteoblastic (bone forming) or osteoclastic (bone degrading) lesions, of which the former are more common in CaP patients (35, 36). Both types of lesion result in high levels of morbidity with symptoms that include severe pain, bone fractures, spinal cord compression, and eventual paralysis or death (35, 36). A model of CaP growth in bone is essential for studying the efficacy of novel therapeutics. The models, including tail-vein, intracardiac, and orthotopic injection resulting in a low incidence of osseous metastasis formation, are complex, or do not adequately reflect the human disease (37). In contrast, intratibial inoculation results in a high incidence of tumor establishment in bone is easy to perform, and results in tumors that are radiologically and histologically similar to those encountered clinically (25).

In the present study, we chose intratibial injection into NODSCID as our therapeutic model and first showed 100% take rate 2 weeks post–cell inoculation. The therapeutic effect of MTAT was also evaluated in PC-3 grown intratibially at 7 days postinoculation. Our findings indicate that a single systemic injection of MTAT at 296 and 592 MBq/kg strongly inhibited tumor progression of CaP cells in bone for at least 3 to 6 weeks. The take rate of PC-3 tumors assessed 6 weeks post–cell inoculation was ∼60% in mice receiving a low dose (296 MBq/kg) and 50% in those receiving a high dose (592 MBq/kg) of MTAT, compared with 100% in control groups (see Supplement Table S3). The tumor burden in mice with bone tumor growth was also reduced to 40% to 50% compared with the controls. By radiographic examination, tumors in bone in this model can be seen 2 weeks after cell inoculation and can then be followed using a semiquantitation score. These scores were smaller (0.6-0.8) in MTAT-treated groups than in control groups (0.8-3.6). Moreover, tumor in MTAT-treated (high dose) mice was significantly different from that in control groups at 4 and 6 weeks (P < 0.05). These results suggest that MTAT could target 30% to 40% of micrometastases in bone tumors and regress tumor growth for at least 6 weeks in a dose-dependent fashion. The growth of PC-3 tumors in tibia is dependent on the interaction between the microenvironment in bone and tumor cells, although the exact mechanisms involved remain incompletely understood. At 7 days post–cell inoculation, tumor cell clusters are still small and may be in micrometastatic stages. MTAT could kill such micrometastatic clusters of cells expressing heterogeneous tumor-associated antigens, whereas larger tumors may only undergo partial regression due to the delivery of an effective dose of MTAT.

The response of tumor growth and lymph node metastases in three models to MTAT was found to be specific, highly dependent on antigenic expression and the concentration of radioactivity. At the end of experiments, most of the tumor antigen expression in the lesions disappeared after treatment with MTAT, whereas only few scattered debris were found positive in these areas. These results suggest that MTAT can specifically target tumor antigen–positive cancer cells. After binding cell surface antigens, the high energy α-particles may be delivered to cancer cells and cause cell death. Macklis et al. (38) postulated that a traverse of at least four α-particles through the nucleus is sufficient to kill a cell. Surface antigen-bound 213Bi-BLCA-38 can deliver the α-particles and kill adjacent CaP cells as shown in our previous study using 213Bi-BLCA-38 conjugate to target antigen-positive CaP cells (10). We have also shown that 213Bi-PAI2 and 213Bi-C595 conjugates alone can efficiently target and kill antigen-positive CaP cells (10). 213Bi-BLCA-38 is not internalized after binding,7

7

Unpublished data.

although this does not affect the ability of the α-conjugates to deliver a toxic payload (10) or to deliver a toxin that is independent of internalization (39), whereas 213Bi-PAI2 AC is internalized through the interaction of protein PAI2 with the uPA/uPAR complex, which leads to internalization of the ternary complex in the PC-3 cell line (18). It is still not known whether or not MAb C595 is internalized after binding CaP cells. However, many factors, including antigen affinity and antigen density, play important roles in the killing of targeted antigen-positive cells. For example, after binding cell MUC1 antigen, 213Bi-C595 may form 213Bi-C595-MUC1 complexes at the cell surface membrane, emitting α-particles that can kill CaP cells by causing double-DNA strand breaks (40). Alternatively, surface-bound 213Bi-C595-MUC1 complexes may be internalized by the cell with increased cell killing efficiency, as suggested in 213Bi-J591–mediated killing of CaP cells (7).

With MTAT, different α-conjugates may act on cancer cell clusters (micrometastases) in lymph nodes or at transit in a concomitant or synergistic manner, so that cells expressing different antigenic targets will sustain bombardment with many disintegrations leading to cell death; treatment with one targeting agent to an antigen-negative CaP cell could result in a sublethal dose. In two CaP animal models (s.c. and orthotopic), it is very difficult to evaluate cell killing in micrometastases (lymph nodes or cancer clusters at transit). However, the histologic changes in primary tumors could be easily assessed. We found scattered targeted lesions in both models with destruction of tissue structure and cell debris (necrosis) after 1 week's treatment with MTAT, and similar targeted lesions were also found in the intratibial lesions after 5 weeks' treatment with MTAT, whereas the nontargeted areas showed “normal” tumor structure. The tumors treated with nonspecific α-conjugates also showed untargeted tissue structure. These results suggest that MTAT can specifically kill CaP cells in xenografts with a limited range because of short half-life, short distance, and slow antibody penetration kinetics of MTAT. For radiation induced renal toxicity, 8 weeks should be considered as short-term toxicity. We have evaluated the histology at 8 weeks in our animal models and did not find any renal damage with either 592 or 296 MBq/kg. Given that our cytoxicity studies suggest that repeat doses may be well-tolerated in NODSCID mice, it will be interesting to test multiple doses of MTAT or single α-conjugates as controls in these models for long-term CaP remission in the future studies.

As described above, treatment with MTAT could reduce the volume of tumor xenografts, delay tumor growth, and prevent lymph node metastases. Although the potential mechanisms for this remain unclear, we conducted preliminary studies to determine whether apoptosis were involved, given our recent demonstration that 213Bi-immunoconjugates could induce a high percentage of TUNEL-positive cells in single monolayer CaP cells (10, 20); this was in contrast to studies on CaP spheroids where only a low percentage of apoptotic cells was found after MTAT, suggesting that apoptosis was not the only mode of cell death. However, apoptosis was not the only mode of cell death observed, consistent with the suggestion that the cell death in single CaP cells and spheroids exposed to α-immunoconjugates can occur via different pathways (9, 41). Many genes and interdependent pathways are involved in the cellular response to ionizing radiation (42). The simultaneous activation of several genes and different pathways can lead to cell death and/or cell proliferation. Another possible mechanism for cell killing in tumors may be a “bystander effect” specifically by α-particles (43). This effect is related to the release of enzymes and factors (e.g., cytokines) from directly hit cells that may have a significant biological effect on the surrounding cells (44). Whether MTAT also initiates this bystander effect in tumors remains to be determined. The exact pathways involved in cell death in CaP xenografts after MTAT clearly requires further study.

In summary, we have established three CaP models in NODSCID mice using the PC-3 cell line and showed that 100% of CaP xenografts and cancer cell clusters tested were positive for at least one of the selected target antigens. Furthermore, MTAT with a cocktail of test α-conjugates selectively regressed cancer cell growth and prevented cancer metastases in three animal models in a dose-dependent manner, and could effectively target lymph node metastases with an activity of 592 MBq/kg. Consequently, MTAT may be a potent therapeutic agent against micrometastatic CaP in late stage hormone refractory disease, overcoming the problem of heterogeneous expression of targeted antigens. The potential of toxicity of 213Bi is myelosuppression and radiation nephritis. However, at the 8-week end point, histology did not reveal any renal damage after either 592 or 296 MBq/kg.

No potential conflicts of interest were disclosed.

Grant support: Department of Defence Prostate Cancer Research Program (W81XWH-04-1-0048; Y. Li) and Cancer Institute NSW Career Development Fellowship (Y. Li).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Professor Alan Perkins (Nottingham University, Nottingham, UK) for providing MAb C595; Professor Andrew Collins (University of New South Wales, Sydney, NSW, Australia) for providing MAb A2; the Minomic Pty Ltd (Sydney, NSW, Australia) for providing MAb BLCA-38 and PAI2 Pty Ltd (Sydney, NSW, Australia) for providing Human recombinant PAI2 (47 kDa); Dr. Michele C. Madigan, School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia for the help in photograph preparation; and Professor J Kearsley, Director of Radiation Oncology, Cancer Services Division, St George Hospital, Sydney, Australia, for the ongoing support.

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